The practical utility of genetic engineering often depends on introducing inheritable genetic traits into organisms. To achieve this goal, foreign DNA must be stably integrated into the DNA of the host organism. Stable integration of foreign DNA into host DNA is often referred to as "transformation" of the host cell (or genome of the cell).
Genetic transformation in higher eucaryotes is often accomplished through the use of viral vectors which rely on stable integration in the host genome as part of their replicative cycle. Retroviruses are one of the few animal viruses that depend upon integration for replication. A number of retroviral vector systems are currently available to mediate transformation of animal genomes. Such systems utilize one or more vectors, at least one of which contains the portion of the retroviral genome responsible for integration of the viral genome into the host genome.
Integration of retroviral DNA requires a virus-encoded enzyme, the integrase (IN), which is encoded by the viral pol gene and carried within the virus particle. (For a review of the retroviral enzymes, including integrase, see Katz & Skalka, Ann. Rev. Biochem. 63: 133-173, 1994). Integration also requires cis-acting sequences at the ends of linear viral DNA. Integration is site-specific with respect to the viral DNA (it occurs at the linear ends), but appears to be nearly random with respect to host DNA.
Biochemical and genetic experiments indicate that integration takes place through two steps. First, IN nicks the viral DNA two nucelotides from the 3' ends of each DNA strand (referred to as the "processing" reaction). This nicking exposes the highly conserved CA dinucleotides, usually located two nucleotides from the 3' end of each strand. The new 3'-OH ends of each viral DNA strand are then joined to the host DNA in a second reaction (referred to as the "joining" reaction). The joining reaction is believed to proceed by a direct attack mechanism whereby the 3'-OH ends of viral DNA strands attack host DNA phosphates that are staggered by 4-6 base pairs. The simplest model for IN function is one in which a single monomer is bound to each end of viral DNA and each monomer is capable of binding viral DNA and host DNA simultaneously.
Both processing and joining activities can be assayed in vitro using short synthetic DNA substrates that mimic the single ends of retroviral DNA (see Katz & Skalka, 1994, supra). Both reactions are thought to be catalyzed by a single active site, due to the chemical similarity of the two reactions and the general inability to biochemically separate the two activities by mutagenesis.
IN is the only viral gene product required for integration of viral DNA into a host genome. For this reason, IN may be used to advantage to facilitate genetic transformation of eucaryotic cells. However, its utility is limited due to its lack of sequence specificity with respect to the host DNA. That is, IN-catalyzed integration can occur essentially at random in the genome, which could result in activation or deactivation of host genes essential for cellular function. Thus, it would be a significant advance in the art of genetic transformation to develop retroviral integrases capable of site-specifically catalyzing integration of foreign DNA into a pre-determined location in the host genome.
It is an object of the present invention to provide modified retroviral integrases capable of enhancing the integration reaction and catalyzing integration of foreign DNA at a selected target location in a host genome. It is further an object of the present invention to provide retroviral vectors that encode such modified integrases, and which also contain the foreign DNA to be inserted into the host genome.